Disrupted proteostasis is a defining feature of neurodegenerative diseases (ND) including Alzheimer and Parkinson disease, and is characterized by the appearance in brain cells of abnormal ubiquitin conjugates and nonnative protein aggregates. Protein aggregation is normally suppressed by the proteostasis network (PN) highly interconnected system that integrates the regulation of gene expression, signaling pathways, molecular chaperones and protein degradation systems. The long-term goal of the proposed research is to understand how the PN senses and responds to specific proteome insults, particularly as they apply to ND pathogenesis. Previous studies of the PN have relied on the application of heavy-handed stressors such as heat shock that simultaneously perturb the entire proteome, or expression of disease related aggregation-prone proteins that are toxic and accumulate slowly and asynchronously. In neither case has it been possible to deconvolve cause from effect in PN response. The central hypothesis of the proposed research is that misfolding of a single protein can initiate an immediate and precise, stereotypic response by the PN by competing with normal client proteins for key limiting PN machinery components. To test this hypothesis, we will exploit AgDD-GFP, a small globular protein that is soluble and stable in the presence of its engineered, biologically inert, cell-permeant small molecule ligand, but which rapidly (<30s) and simultaneously unfolds and aggregates upon ligand removal. Three specific aims are proposed using unbiased, novel biochemical, proteomic and genetic techniques to systematically and comprehensively identify the key players in suppressing and responding to AgDD misfolding and aggregation and to define the precise temporal sequence with which these proteins respond. Aim 1 will comprehensively identify changes to the cellular ubiquitinome that accompany AgDD-GFP (and its non-aggregating variant, DD-GFP) unfolding and aggregation and a recently developed model of inducible alpha synuclein aggregation in primary neurons.. Aim 2 will use proximity labeling to identify interacting proteins and the temporal sequence with which they bind to AgDD-GFP or DD-GFP and hits will be validated in primary neurons. Aim 3 will assess the effects of AgDD-GFP (and DD-GFP) unfolding and aggregation on ribosome modification and protein synthesis. The proposed research is significant because it will comprehensively define the cellular response to the type of PN insult that precedes and perhaps initiates the PN collapse that underlies cellular pathogenesis in many ND. The proposed research is innovative because it uses novel approaches to allow, for the first time, interrogation of protein unfolding and aggregation to reveal with unprecedented molecular and spatiotemporal resolution, how the PN responds to a defined insult.